Introduction

Charophyceae is macroscopic algae, habit in both fresh and brackish water, upright stem like structure with branches, in addition to whorl of secondary branches ¹⁵. These members are in close relation to higher plants due to the presence of similar pigmentation and reproductive structures ⁴. They are used as research models in biotechnology, genetic engineering, biochemistry, cell cycle and physiology ⁵. Algae have the great importance in ecological and economical context. Hence the availability of systematic details, diversity and distribution is essential for future research work.  Systematics deals with identification of plant and evolutionary relationships ³. They are studied by the classical and molecular methods.  In classical method the plants are identified based on the morphological characteristics and inmolecular method plant are identified with the DNA sequence ¹. Identification of organism through molecular phylogenetic study can be done in short gene from a uniform locality on a genome. Each organism has different type of DNA barcode, so it has the great importance in the taxonomy, evolution and phylogenetical arrangement ². The first step of the Molecular phylogenetic studies is to select the suitable primers, amplified the barcode region PCR. In plant species which show similar morphology, differences can be identified through molecular phylogenetic study ¹². Sometimes it is difficult to identify the Chara speciesin which do not bear sex organs ¹⁵. In such conditions the Molecular phylogenetic study helps in the identification of Organism. Anyone who has the Basic knowledge on DNA technology is adequate to identify the organism. Molecular phylogenetic study is not the only solution for all taxonomical problems, but it provides the effective nomenclature ¹⁰ and identification for the unknown or un recognized species. The organisms which are not identified in the classical methods can be identified by the molecular phylogenetic study.

Material and Methods

Collection and Culture of Chara species

Chara sample was collected from the fresh water streams of Talakona region, Chittoor district, Andhra Pradesh. The sample was isolated from other associated algae, cleaned thoroughly with the water ¹⁹.Chara sample was cultured in in vivo condition and named as CH9.

Molecular Phylogenetic

DNA was isolated from the samples with DNeasy plant Mini Kit(Quiagen, Hilden, Germany). 1.0 % agarose gel was used for quality assessment, a high-molecular weight single band  DNA has been identified. 

CDMFP and CDMRP primers were used for amplifying the fragments of 18S rRNA gene. 700 bp a single discrete PCR amplicon band identified in the Sample when resolved in agarose gel. CDMFP and CDMRP primers are carried out the DNA sequencing reaction. Forward and reverse PCR amplicon was used in BDT v3.1 Cycle (sequencing kit on ABI 3730xl)on Genetic analyzer for eliminating contaminants.

Aligner software used for generating Consensus sequence of 18S rRNA from forward and reverse sequence data. The 18S rRNA gene sequence was used to carry out BLAST with the ‘nr’ database of NCBI GenBank database. Ten sequences are selected based on the Maximum identity score and multiple alignment software program Clustal W was used for alignment. Ribosomal Database Project was used for generating distance matrix. Molecular Evolutionary Genetics Analysis 6 software was used for constructing the phylogenetic tree.

Results

Morphological Characters    

Plant is 30 cm long, 620 µm wide and 6-9 whorls branch lets,incurved, terminal cell with 3 projections.

Collection number: Talakona surrounding areas.

Distribution: First report from Andhra Pradesh [6] 

Figure 1: Chara foeitda.   Click here to view figure

Molecular Phylogenetic

Charaspecies shows 91.16% homology to the Charafoeitda. So, Chara sample was identified as Chara foeitda

Maximum Likelihood method (Tamura-Neimodel) are used for the evolutionary history ^16,17  CH9 sample tree shows highest log likelihood at (-1309.77). 11 nucleotide sequences are involvedin analysis and Codon positions included were 1st+2nd+3rd+Noncoding.In Chara foetida total of 737 positions in the final data set. Evolutionary analyses were conducted in MEGA 6.

Figure 2: Genomic DNA and Amplicon QC data    Click here to view figure

CDMRP Primer

Figure 3: CDMFP Primer   Click here to view figure

Table 1: Sequences producing significant alignment

Figure 4: Sanger Seq Chromatogram data file.    Click here to view figure

Table 2: Distance matrix

Figure 4: Phylogenetic Tree   Click here to view figure

Discussion

The taxonomical studies based on the morphology sometimes may leads to the misidentification ¹¹. To overcome to this, molecular approaches are used. The 18s RNA, matK, ITS2 and rbcl genes are most widely used and effective methods in Molecular phylogenetic ¹³.

The class Charophyceae members are mostly benthic in nature, complex structure. The identification of these members is difficult due to the presence of phenotypic plasticity. The present study identified the macroscopic algae Chara with molecular phylogenetic technique. Chara species  is identified by sequencing of 18s RNA gene analysis, namely Chara foeitda. The sample code CH9 shows 91.16% similarity to the Chara foeitda.

Monique Turmelet al (2006) studied the atpB, rbcl, 18S rRNA and mitochondria (nad5) of Chara vulgaris and it showed closest relation to the green plants of land ¹⁸. Hidetoshi identified the Chara globularis with molecular phylogenetic based on the rbcl gene sequencing ⁷. Schneider et al identified the 14 species of Chara with barcodes like ITS2, Mat K and rbcl genes from Europe ¹⁴. Jacek& Michal studied the Chara globularis Var. tenuispina and Chara globularis phylogenetic relationship ⁸. Based on the atpB, matK and rbcl sequences and identified C. tenuispina distinct species from Chara globularis ⁹. The present study is one of the pioneer attempt to the molecular phylogenetic for  Charophyceae of India.

Conclusion

Chara species are identified as Chara foetida based on the molecular phylogenetic. For the identification 18S rRNA sequence was used to carry out BLAST with the ‘nr’ database of NCBI GenBank database. First ten sequences were selected based on maximum identity score using multiple alignment software program Clustal W. Ribosomal Database Project was used for generating distance matrix and Molecular Evolutionary Genetics Analysis 6 Software was used for constructing the phylogenetic tree. Based on the maximum identifying score Chara species was identified as Chara foetida.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

References

1. Akash P, Bakhtiyar AS, Bharat G, Pankaj P, Beena P, Rbcl Marker based approach for molecular identification of Arthrospira and Dunaliella isolates from non- Axenic culture, Journal of Genetics and Genetic Engineering, 2018, 2:24-34. https://www.researchgate.net/publication/328118071_ Rbcl_Marker_Based_Approach_for_Molecular_Identification_of_Arthrospira_and_Dunaliella_Isolates_ from_Non-Axenic_Cultures
2. Alberts B,  Johnson A, Lewis J, Molecular Biology of the Cell. Garland Science; Studying Gene Expression and Function, Garland Science, New York, 2002, 1-50, https://www.ncbi.nlm.nih.gov/ books/NBK26818/
3. Braun .A, Esquissemono graphique du genre Chara. Ann. Sci. Nat., sér., 1834, 1: 349-357.
4. David. D, Iben.S, Zoe .P, Charophytes: Evolutionary Ancestors of plants and Emerging models for plant research, Frontiers, USA, 2017, 1-5, doi: 10.3389/fpls.2017.00338
   CrossRef
5. David. D, Zoe.P, Iben.S, Charophytes: Evolutinorygaints and emerging model organisam, Frontiers, USA, 2016, 6-10, https://doi.org/10.3389/fpls.2016.01470
   CrossRef
6. Gupta RK, Algae of India Vol.2. Achecklist of Chlorophyceae, Xanthophyceae, Chryophyceae and Euglenophyceae, Botanical survey of India, Kolkata, 2012, 9-331.
7. Hidetoshi Sakayama, FumieKasai, Hisayoshi Nozaki, Makoto M. Watanabe, Masanobu Kawachi, Mikao Shigyo, Jun Nishihiro, Izumi Washitani, Lothar Krienitz and Motomi Ito, Taxonamic re-examination of Chara  globularis (Charales, Charophyceae) from Japan based on oospore morphology and gene sequences and their description of c. Leptospora Sp.Nov, Journal of Phycology, 2009, 45: 917-927, https://doi.org/10.1111/j.1529-8817.2009.00700
   CrossRef
8. Jacek U, Hidetoshi S, Taxonomical analysis of closely related  species of CharaL.section Hartmania (Streptophyta: Charles ) based on morphological and molecular data, Fottea, 2017A,  17:222-239, DOI:10.5507/fot.2017.004
   CrossRef
9. Jacek U, Michal C, Taxonomical status of Chratenupina A. Br. Based on LM morphology, matK, atpB, and rbcl of cpDNA sequences, Fottea, 2017B, 17:20-33, DOI: 10.5507/fot.2016.011
   CrossRef
10. Jahn C.E.,  Amy O. C,  David K. W. Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation, Journal of Microbiological Methods, 2008, 75:318–324, doi.org/10.1016/ j.mimet.2008.07.004.
    CrossRef
11. Karan T, Erenler R, Altunar Z, Isolation and Molecular identification of some blue green algae (cyanobacteria) from Fresh water sites in Tokat Province Turkey, Turkish Journal of Agriculture – Food Science and Technology, 2017, 5:1371-1378, doi.org./10.24925/turjaf.v5i11.1371-1378.1470.
    CrossRef
12. Lorenz TC, Polymerase chain reaction: basic protocol plus trouble shooting and optimization strategies,  Journal of visualized experiments 2012, 63, doi:10.3791/3998.
    CrossRef
13. Monique T, Christian O, Claude L The Chloroplast genome sequence of Chara vulgaris Sheds new light into the closet green algal relatives of land plants, Molecular biology and evolution, 2006 , 23: 1324-1338, doi:10.1093/molbev/msk018.
    CrossRef
14. Prakash S, Sanjay K.V, Identification and characterization of green microalgae, Scenedesmus Sp. and  Auctodesmus obliquus MCC33 isolated from Industrial polluted site using morphological and molecular markers, International Journal of Applied Sciences and Biotechnology, 2018, 5: 415-422, doi.10.3126/ijasbt.v5i4.18083.
    CrossRef
15. Schneider SC, Susanne, Rodrigues A, Anuar R,  Therese MTF, Ballot A, NDNA barcoding the genus Chara: Molecular evidence recovers fewer taxa than the classical morphology approach, Journal of Phycology, 2015, 51: 367-380, doi: 10.1111/jpy.12282.
    CrossRef
16. Subramanian, Indian Charophyta, MJP Publishers, Chennai, 2018,  45- 168.
17. Tamura K., Nei M, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 1993, 10:512-526, doi: 10.1093/oxfordjournals.molbev.a040023.
    CrossRef
18. Tamura K., Glen S, Daniel P, Alan F, Sudhir K, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 2013,  30: 2725-2729, doi: 10.1093/molbev/mst197.
    CrossRef 
19. Zulkarnain C Fadjria, N.,  Armaini, Armaini, Zainul, Rahadian, Isolation and molecular identification of Fresh water microalgae in Maninjau lake West Sumatra, Der Pharmacia Lettre, 2016 , 8:177-187, https://www.researchgate.net/publication/316888807_Isolation_and_molecular_identification_of_freshwater_microalgae_in_Maninjau_Lake_West_Sumatra.